The aim of the proposed research is to elucidate the quaternary structure and mechanism of action of the enzyme hydrogenase. Pure hydrogenase from Chromatium, Proteus vulgaris, and Escherichia coli will be studied to establish the structure of the active iron-sulfur site and the nature of its reaction with substrate and inhibitors. The different forms of the enzyme will be identified and differentiated by activity and EPR spectra and will establish the number of electrons accepted by the enzyme from the substrate. The same techniques will be applied to the interaction of the enzyme with the inhibitors oxygen, carbon monoxide, and nitric oxide. Experiments will be carried out to demonstrate conclusively the formation of an enzyme hydride intermediate. This research will also investigate the regulation of hydrogenase synthesis in microorganisms which will establish the role of the enzyme in microbial physiology, define conditions for induced synthesis of the enzyme at high levels, and permit mapping of the hydrogenase gene and its transfer to organisms lacking the enzyme. Approaches are outlined for isolation of mutant forms of hydrogenase with altered properties. Of particular interest is the isolation of an oxygen insensitive enzyme which retains full catalytic activity in the presence of oxygen.